| 慢病毒载体法建立乳腺癌GFP-ST3稳定细胞株 |
| 投稿时间：2015-01-22 修订日期：2015-02-03 点此下载全文 |
| 引用本文：崔红霞,王明,赵学梅,邹宇,张奇.慢病毒载体法建立乳腺癌GFP-ST3稳定细胞株[J].医学研究杂志,2015,44(9):57-59,62 |
| DOI：
10.11969/j.issn.1673-548X.2015.09.015 |
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| 基金项目:黑龙江省普通高等学校青年学术骨干计划基金资助项目(1253G067) |
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| 中文摘要:目的 慢病毒载体法建立乳腺癌GFP-ST3稳定细胞株。 方法 采用PCR扩增α2,3-ST全长基因片段,克隆至慢病毒载体进行测序分析,将重组慢病毒载体转染人乳腺癌细胞,嘌呤霉素抗性筛选,经荧光显微镜观察、实时定量PCR、Western blot检测α2,3-ST的表达。 结果 成功构建了慢病毒载体pGLV5-H1-GFP-ST3,体外转染乳腺癌细胞后,荧光显微镜下可见绿色荧光蛋白的表达,α2,3-ST mRNA表达和蛋白水平均明显升高(P<0.05),获得稳转细胞株。 结论 成功构建了绿色荧光蛋白为报告基因的α2,3-ST慢病毒载体,并在MDA-MB-231中稳定表达,为进一步研究乳腺癌细胞膜连α2,3-唾液酸水平增高与其侵袭和转移潜能的调控提供实验模型。 |
| 中文关键词:α2,3-ST 绿色荧光蛋白 转染 表达 |
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| Establishment of Human Breast Cancer Cells with Stable Expression of GFP-ST3 Via Lentivirus Vector |
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| Abstract:Objective To establish the human breast cancer cells with stable expression of GFP-ST3 via lentivirus vector. Methods The human α2,3-ST cDNA was obtained and amplificated by polymerase chain reaction.Then the gene was incorporation into pGLV5-H1-GFP plasmid. The recombinant vector pGLV5-H1-GFP-ST3 was transfected into breast cancer cells. A stably transfected cell line was established using puromycin resistance selection. The expression of α2,3-ST was observed using fluorescence microscope and estimated by real time PCR and western blot. Results The recombinant plasmid pGLV5-H1-GFP-ST3 was successfully constructed. After transfection into breast cancer cells, green fluorescence was observed. Analysis displayed that the level of α2,3-ST mRNA and protein was significantly increased in MDA-MB-231 cells. Conclusion The α2,3-ST lentivirus vector pGLV5-H1-GFP-ST3 that can express α2,3-ST in MDA-MB-231 cells is constructed successfully and can be used to study the correlation of breast cancer metastatic potential with the overpression of α2,3-ST gene. |
| keywords:α2,3-ST Green fluorescent protein Transfection Expression |
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